JP5419455B2 - Use of lactic acid bacteria for the treatment of autoimmune diseases - Google Patents

Description

  The present invention relates to the use of at least one Probiotic bacteria strain selected from lactic acid bacteria for the manufacture of a pharmaceutical composition for the treatment and / or prevention of autoimmune diseases.

  Probiotic bacteria are defined as living microorganisms, and have a beneficial effect on the host at the right dose. Lactic acid bacteria and bifidobacteria are the most frequently used bacteria for probiotic products. These bacteria are generally safe, as are probiotic products based on these bacteria. It is not pathogenic to any age and is not pathogenic to immunocompromised individuals. The intake of various probiotic bacteria has shown clinical benefit in various physiological and pathological situations. The most obvious effect has been shown in diarrhea caused by antibiotic treatment and rotavirus infection. In addition, after ingestion of probiotic bacteria, good clinical effects have been shown for inflammatory bowel disease, atopic dermatitis, and hypercholesterolemia. The mechanism by which probiotic bacteria contribute to these clinical improvements is unclear. Similar to in vitro and in vivo studies in animals, in vitro studies from humans have shown that various lactic acid bacteria affect innate and acquired immunity in various ways.

  Clinical studies have shown mainly stimulation of the innate cell immune system, enhancement of humoral immune responses to natural infection, and enhancement of oral or systemic immunity. Regarding the innate immune system, increased phagocytic activity of polymorphonuclear cells (PMN) and increased tumor attack activity of NK cells have been reported. To our knowledge, there are no clinical studies showing the effects on the specific cellular immune system after ingestion of probiotic bacteria.

  In accordance with the present invention, the effects of lactic acid bacteria and gram-negative bacteria P. lundensis on the innate immunity and acquired immune system after daily ingestion were thoroughly investigated. Interestingly, specific cellular immune system activation was observed in subjects receiving L. plantarum, and the same was true in subjects receiving L. paracasei. It has been shown. Furthermore, in subjects who received various lactic acid bacteria species, an immune enhancing effect on innate immunity was observed, such as an increase in NKT cell population and enhancement of phagocytic activity. P. rundensis, a gram-negative bacterium, had no effect and did not affect any other measured immune parameters as the experiments described here.

  In Western society, there is a problem with autoimmune diseases and autoimmune related diseases in which the individual's immune system attacks himself with his own mistakes and the individual becomes seriously ill. Autoimmune diseases affect connective tissue and also affect many other parts of the body, such as certain organs such as skin, nerves, brain, lungs, kidneys and joints. An example of an autoimmune disease in the nerve or brain is multiple sclerosis, and an example in the skin is psoriasis. Autoimmune diseases take many different forms, and there are many treatments for them. The treatment depends on the type of disease and the organ affected.

  Within the skill of the art there is a need to treat or alleviate the symptoms of autoimmune diseases as well as provide prophylactic treatment before the disease develops. These problems are the subject of the present invention which will become more apparent below.

SUMMARY OF THE INVENTION The object of the present invention is to provide at least one Probiotic bacteria strain selected from lactic acid bacteria for the manufacture of a pharmaceutical composition for the treatment and / or prevention of autoimmune diseases. Is the use of.

  Another object of the present invention is a method for treating an autoimmune disease, wherein at least one probiotic bacterial strain selected from lactic acid bacteria is administered to an individual.

  In an embodiment of the present invention, at least one probiotic bacterial strain selected from lactic acid bacteria is multiple sclerosis (MS), allergy, psoriasis, rheumatoid arthritis, Crohn's disease, ulcerative colitis, type I diabetes, inflammatory Used in the manufacture of a pharmaceutical composition for the treatment of an autoimmune disease selected from bowel disease or organ specific autoimmunity such as systemic lupus erythematosus.

  Although not specifically mentioned, any other autoimmune disease in which the probiotic bacterial strain has an effect (which can be prevented or treated) is within the scope of the present invention.

  As used herein, the term “treatment and / or prevention” refers to prophylactic treatment in an individual, ie, treatment with a probiotic bacterium to prevent the disease is initiated before the disease develops, and Includes treatment of diseases already developed in the individual. In the latter case, for example, symptom relief is expected, or the patient's general condition goes better, or the patient is cured from the disease. That is, the individual can be an individual who is not at risk of developing an autoimmune disease or a patient who already has the disease.

  Lactic acid bacteria used according to the present invention include Lactobacillus plantarum, Lactobacillus rhamnsosus, Lactobacillus fermentum, Lactobacillus paracasei, Lactobacillus paracasei, Lactobacillus paracasei, Although it is selected from the group consisting of gasseri (Lactobacillus gasseri), it is not limited to them.

  Other probiotic bacterial strains can be used according to the present invention than the probiotic bacterial strains specifically disclosed herein. Those strains are within the scope of the present invention as long as they provide the expected effect, ie, alleviation of symptoms of autoimmune disease or prevention of autoimmune disease.

  The Lactobacillus plantarum used according to the present invention is Lactobacillus plantarum 299, DSM 6595, Lactobacillus plantarum 299v, DSM 9843, Lactobacillus plantarum HEAL 9, DSM 15312, Lactobacillus plantarum HEAL 19 , DSM 15313, Lactobacillus plantarum HEAL 99, DSM 15316, but not limited thereto.

  The Lactobacillus paracasei used in accordance with the present invention may be selected from the group consisting of, but not limited to, Lactobacillus paracasei 8700: 2, DSM 13434, Lactobacillus paracasei 02A, DSM 13432.

  The Lactobacillus gasseri used in accordance with the present invention may be selected from, but not limited to, Lactobacillus gasseri VPG44, DSM 16737.

  In another embodiment of the invention, at least two probiotic strains are used for the treatment and / or prevention of said autoimmune diseases. The at least two strains are preferably selected from lactic acid bacteria from Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus fermentum, Lactobacillus paracasei, and Lactobacillus gasseri.

  In this embodiment where at least two probiotic strains are used for the treatment of interest, it is contemplated that the two strains are administered sequentially or simultaneously. That is, the strains may be administered in a mixed form as one composition, or may be administered independently and sequentially as different compositions.

  Furthermore, in an embodiment of the present invention, the pharmaceutical composition is a liquid formulation or a solid formulation. The solid preparations are tablets, suction tablets, sweet, chewing tablets, chewing gum, capsules, sachets, powders, granules, coated particles and coated tablets, enteric tablets and enteric capsules, The liquid formulation is selected from the group consisting of oral solutions, suspensions, emulsions and syrups.

  Still further, in embodiments of the present invention, the composition may include a carrier material. The carrier material is independently selected from the group consisting of oatmeal meal, lactic acid fermented food, resistant starch, dietary fiber, carbohydrate, protein and glycated protein, but is not limited thereto.

  In a further embodiment of the invention, the pharmaceutical composition is selected from, but not limited to, medical foods, functional foods, dietary supplements, nutritional products, food preparations. Therefore, the term “pharmaceutical composition” as used herein does not necessarily mean a pharmaceutical composition in the usual sense, like a pharmaceutical product, but a medical food, functional food, dietary supplement. , Nutritional products, products in the area of food products. In the case of said food products, the food products may be selected from but not limited to beverages, yogurt, juices, ice creams, breads, biscuits, cereals, health bars and spreads.

  Thus, the use of the composition according to the invention can be very beneficial in the sense that it can be used prophylactically, ie before an autoimmune disease develops. The pharmaceutical composition used need not be a medicinal product in the usual sense, and may be a health supplement or a functional food, so that the normal healthy person can take the composition of the present invention prophylactically. Very convenient.

In an embodiment of the invention, each of the aforementioned strains in the composition is about 1 × 10 ⁶ to about 1 × 10 ¹⁴ CFU, preferably about 1 × 10 ⁸ to about 1 × 10 ^12, more preferably 1 × 10 ^9. Or present in an amount of 1 x 10 ¹¹ .

  The pharmaceutical composition according to the invention may also contain other substances well known to those skilled in the art such as inert vehicles or pharmaceutically acceptable adjuvants, carriers, preservatives and the like.

Example
Example 1
Subjects and clinical trial criteria
57 clearly healthy volunteers aged 18 to 55 years (average 26 years) were selected for a blinded placebo-controlled study. Subjects were randomly assigned to 8 groups, Gram-positive bacteria, L. plantarum 299v (n = 7), L. plantarum Heal (L. plantarum Heal) 19 (n = 7), L. fermentum 35D (n = 7), L. paracasei 8700: 2 (n = 7), L. gasseri VPG 44 (n = 7), L L. rhamnsosus 271 (n = 7) or Gram-negative P. Lundensis n = 7) or placebo (n = 10). Bacterial doses were 10 ¹⁰ bacteria / day for lactic acid bacteria and 10 ⁹ bacteria / day for P. rundensis. The subject group received 1 g of skim milk powder. Depending on the group, the study was conducted for 6 or 9 weeks. Of these, 2 weeks are drug holiday, the active study period is 2 or 5 weeks, and the rest is a 2 week follow-up period (Figure 4). Each subject was given a list of products containing probiotic products that should not be consumed during all study periods. Peripheral blood samples were collected from the subject's vein a few times on the 0th, 14th, and 35th days. Diaries that recorded the subject's complaints of side effects, health status, and confirmation of the research product consumed were recorded during the trial.

Flow cytometry Analysis of lymphocyte phenotype in whole blood was performed by flow cytometry. The following anti-human monoclonal antibodies were used as surface markers for various cell populations. CD3 FITC (SK7), CD4 APC (SK3), CD8 PerCP (SK1), CD19 PerCP (SJ25C1), CD56 PE (MY31), CD16 PE (B73.1), and CD5 FITC (L17F12). The following anti-human monoclonal antibodies are used to detect various activation and memory markers: CD25 FITC (2A3), HLA-DR PE (L243), CD45RO PE (UCHL-1), CD38 PE (HB7 ), CD27 PE (L128), and CD11b PE (D12). All antibodies were purchased from Becton, Dickinson (Erenboden, Belgium). Whole blood (100 μl) is incubated with antibody (10 μl) in the dark at 4 ° C for 30 minutes, then 2 ml FACS lysing solution (Becton, Dickinson) is added, and in the dark at 20 ° C for 15 minutes Incubated. The cells were washed with 3 ml of FACSFlow, centrifuged at 300 × g for 5 minutes, and resuspended with 200 μl of FACSFlow. Analysis was performed using FacsCalibur (Becton, Dickinson) and CellQuest software.

Phagocytic assay The phagocytic activity of monocytes and granulocytes was measured using PHAGOTEST (registered trademark) (Orpegen Pharma, Heidelberg, Germany) with some modifications according to the manufacturer's specifications. Briefly, 20 × 10 ⁶ FITC-labeled E. coli or S. aureus are added to pre-chilled whole blood (100 μl). Blood cells and bacteria were incubated at 37 ° C. for 10 minutes, and FacsCalibur and CellQuest software were performed.

Calculations Inter-individual changes for various immune parameters were determined by calculating the ratio of individual values obtained on day 14 and day 0, or on day 35 and day 0. These ratios were used for calculation and statistical processing for all groups.

Statistical processing All statistical analyzes were performed using Statview. Mann-Fitney U test was used to compare between different groups.

result
Clinical observation
Of the 57 volunteers, 54 were fully studied. Two were infected and excluded for antibiotic treatment (one in the placebo group and the other in the P. rundensis recipient group). One patient was excluded due to pregnancy on the 16th day (placebo group). Only mild gastrointestinal side effects were reported after ingestion of research products (Figure 1).

The activation markers CD4 ⁺ and CD8 ⁺ T cells, which activate T cells by lactic acid bacteria intake, had individual variability and the baseline (Day 0) was very high. A baseline of the percentage of cells expressing various cell surface markers is shown in FIG. There were no significant differences between the various groups at this time. Since very large inter-individual differences were observed, they were selected to compare the ratio values on day 14 and day 35 compared to day 0 for each individual. All calculated values and their comparisons were made at ratio values (Day 14 / Day 0 and Day 35 / Day 0). Approximately 14 times higher expression of the activation marker CD25 on CD8 ⁺ T cells was observed 14 days after ingestion of the study product containing L. plantarum 299v (p = 0.01) (FIG. 5). In addition, although not significant (p = 0.12), ingestion of L. plantarum 299v also showed strong up-regulation of HLA-DR on CD8 ⁺ cells. In addition, a trend towards CD4 ⁺ T cell activation was observed after ingestion of L. plantarum 299v. Ingestion of other lactic acid bacteria included in this study as well as the Gram-negative bacterium P. rundensis did not activate CD4 ⁺ T cells or CD8 ⁺ T cells. However, ingestion of L. paracasei certainly tended to increase the expression of HLA-DR in CD4 ⁺ T cells (p = 0.18).

Ingestion of lactic acid bacteria induces a memory-type phenotype of CD4 ⁺ T cells
The geometric means of the fluorescence intensity (GMFI) of CD45RO expression on CD4 ⁺ and CD8 ⁺ T cells was compared between groups receiving different study products. As above, the calculated values for the groups were used for comparison based on individual ratio values (Day 14 / Day 0 and Day 35 / Day 0). 35 days after taking the study product containing L. plantarum 299v, the GMFI value of CD45RO in CD4 ⁺ T cells was significantly elevated (p = 0.03). There was also a tendency for CD45RO to increase on CD8 ⁺ T cells after ingestion of L. plantarum (Figure 6). Moreover, ingestion of L. paracasei seems to have a positive effect on the upregulation of CD45RO on CD8 ⁺ T cells' (p = 0.10) (Figure 6)

Effects of research products on various cell populations
Ingestion of L. paracasei increased the percentage of lymphocytes that could be identified as NKT cells (p = 0.06) (Figure 7). CD4 ⁺ T cells, CD8 ⁺ T cells, B cells, B-1 cells (CD19 ⁺ , CD5 ⁺ ), NK cells, granulocytes, relative to the increase or decrease compared to day 0 It could not be confirmed for other cells such as monocytes.

Phagocytic granulocytes and monocytes can be identified on the FSC-SSC diagram. These cells were tested for phagocytic activity of FITC-labeled Gram-negative and Gram-positive bacteria. As shown in Figure 8, received L. plantarum 299v (p = 0.064), L. plantarum Heal19 (p = 0.064), L. fermentum (p = 0.064), or L. paracasei (p = 0.05) Granulocytes obtained from volunteers showed phagocytosis of Escherichia coli, a Gram-negative bacterium, more effectively than granulocytes obtained from volunteers in the placebo group. However, S. aureus, which is a Gram-positive bacterium, did not show a difference between phagocytic groups. No difference in phagocytic activity was detected in monocytes (data not shown).

DISCUSSION The primary task of the immune system reacts quickly and furiously with microorganisms, thus preventing and curing infections. We use a powerful mechanism for killing microorganisms and damage our own tissues. Therefore, it is necessary not to respond to our own organization or to the harmless substances present in its environment. Thus, the immune system develops and maintains tolerance to both our own body components and the food and inhaled proteins. If this fails, it causes a number of diseases. The means to generate specific immune tolerance is an essential task in the immune system.

  Helper T cells play a central role in all immune responses. When helper T cells are activated by specific antigens, divide and mature, they produce various cytokines that act on other types of cells in the immune system, such as cytotoxic T cells and B cells. Helper T cell activation is necessary to create most types of immune responses, including antibody production. Conversely, when helper T cell activation is inhibited, most types of immune responses are paralyzed.

  There are a variety of mechanisms that can help to activate T helper cells and maintain tolerance. One mechanism is to eliminate and have the ability to recognize and react to your own tissue within thymic T cells. However, the exclusion is not complete and further development of specific immune tolerance against foreign antigens is necessary. Otherwise, it will react violently to all types of substances inhaled and ingested, causing extensive inflammation and wasting immune resources.

  The cell type central to maintaining tolerance is regulatory T cells. This cell type expresses CD4 and CD25 on the surface, retains CTLA-4 in the cell, and can be recognized by specific markers such as transcription of nuclear protein Foxp3. Regulatory T cells have the ability to suppress the activation of other T cells when encountering innocuous substances, thus avoiding all types of immune responses that are not required.

The symbol “+” in the text associated with a particular marker such as CD4 ⁺ or CD25 ⁺ means that the marker is expressed on T cells. For example, CD4 ⁺ CD25 ⁺ T cells are T cells that express both CD4 and CD25 markers on their surface. However, nothing is said about the amount of marker expressed, only that it is present. The symbol “++” in the text associated with a marker such as CD4 ⁺⁺ or CD25 ⁺⁺ means that the marker is abundantly expressed on the T cell surface. Regulatory T cells are cells that express a large amount of CD25 on their surface, ie CD4 ⁺ CD25 ^{+ +} T cells. In contrast, CD4 ⁺ CD25 ⁺ T cells are simply activated T cells. Sometimes, like CD4CD25, specific symbols such as “+” and “++” are not used, which means activated cells as well as CD4 ⁺ CD25 ⁺ cells. That is, CD4CD25 is the same as CD4 ⁺ CD25 ⁺ . When discussing regulatory T cells, it is always referred to as CD4 ⁺ CD25 ⁺⁺ cells.

  This blind, placebo-controlled study is unique and is the first to compare the effects on different immune parameters after ingesting different gram-positive bacteria, lactic acid bacteria, or gram-negative bacteria P. runnsis for the first time. Interestingly, none of the immune parameters measured certainly affected P. rundensis after ingestion. In contrast, intake of lactic acid bacteria affected different elements of both the specific and innate immune systems. New findings in this study can be asserted that L. plantarum intake positively affects memory cell activation and induction in T cell populations. There was a strong tendency to up-regulate HLA-DR of cytotoxic T cells and a significant up-regulation of IL-2 receptor α chain (CD25). A trend towards up-regulation for these activation markers was also observed in helper T cells after ingestion of L. plantarum. Activation marker expression causes T cells to start proliferating in response to antigen-specific or non-specific stimuli and activates their effector functions faster than resting T cells Is shown. The mechanism behind the activation of T cells induced by L. plantarum is thought to be through the cells presenting antigens, activated by binding of microbial compounds to Toll-like receptors . Activation of antigen presenting cells makes antigen presentation to T cells more effective. Furthermore, both helper T cells and cytotoxic T cells express various Toll-like receptors, and these cells probably perceive non-specific activation by microbial components and products. Conceivable.

From the similarity of the helper T cell compartment, CD45RO expression appears to mark a memory population in cytotoxic T cells. On day 35 after ingestion of L. plantarum, a significant increase in the expression of this memory cell marker in helper T cells and a tendency for up-regulation of cytotoxic T cells were found. Furthermore, L. paracasei also showed a tendency to upregulate CD45RO on cytotoxic T cells. Compared to naive T cells, CD45RO ⁺ T cells secrete cytokines in a wider range. Moreover, CD45RO ⁺ T cells can proliferate and produce IL-2 when the CD3-TCR complex is stimulated under suboptimal conditions. In contrast, naive T cells require strong CD3-TCR stimulation to perform these functions. The formation of memory T cells is important for the induction of an effective immune response after infection and vaccination.

The innate cellular immune system is also caused by ingestion of probiotic bacteria. Following the intake of L. paracasei, it was demonstrated that the natural killer T (NKT) cell population increased. NKT cells constitute a lymphocyte subpopulation that co-expresses the NK cell marker CD56 and the T cell marker CD3-T cell receptor complex. Studies in humans and mice have demonstrated that NKT cells play a central role in the control of autoimmune diseases such as multiple sclerosis, type I diabetes, and systemic lupus erythematosus. NKT cells also exert effector functions against tumors and viral infections. That is, NKT cells are multifaceted with respect to their functions. In other clinical studies evaluating the effect of probiotic bacteria on immunity, intake of L. rhamnousus HN101 and Bifidobacterium lactis HN019 is NK cells (including NKT cells) K562 cells It has been shown to increase tumor killing activity against In this study, it has also been confirmed that the phagocytic activity of polynuclear cells increases after ingestion of various lactic acid bacteria. Activation of cytotoxic T cells and NKT cells in that immune defense is enhanced against infections and / or tumors that increase the virus as a result of the effects on various immune parameters observed in this study It is estimated that the increase of In vitro findings found that lactic acid bacteria induce the secretion of IL-12 and IL-18 in monocytes, and support the theory that the uptake of these bacteria stimulates cell-mediated activity. In accordance with the present invention, it was concluded that the intake of L. plantarum and L. paracasei has a specific and profound effect on the innate cellular immune system.

Example 2
The goal in this example is to examine the effect on the immune system of giving the same species of lactic acid bacteria for a longer period of time compared to the sequential administration of several (different species) lactic acid bacteria one after the other. is there.

Volunteers were given powdered bacteria that were lyophilized for 14 or 35 days. As a Gram-positive bacterium, Lactobacillus plantarum 299v probiotic bacterium was used alone or in combination with L. rhamnosus, L. fermentum, L. paracasei, L. gasseri. Pseudomonas rundensis was used as a gram-negative bacterium.
The study was conducted in the following groups.
1) Lactobacillus plantarum 299v 35 days 2) L. plantarum 299v 7 days, L. rhamnosas 271 7 days, L. fermentum 35D 7 days, L. paracasei 8700: 2 7 days, L. paracasei VGP44 7 days , Total 35 days (continuous)
3) A mixture of L. plantarum 299v, L. rhamnosus 271, L. fermentum 35D, L. paracasei 8700: 2, L. paracasei VGP44. 14 days in total 4) L. rhamnosas 271 14 days 5) L. Fermentum 35D 14 days 6) L. Paracasei 8700: 2 14 days 7) L. Gasseri VGP44 14 days 8) Pseudomonas rundensis 14 days control group 2) Placebo 35 days control group 1) Placebo 14 days Blood samples were collected on day 0, day 14 and day 35. The amount of helper T cells (CD4 ⁺ ) in which a large amount of CD25 was expressed was determined for each group using the flow cytometry specified in Experimental Example 1 above.

result
On day 14, the increase in CD4 ⁺ CD25 ⁺⁺ T cells was borderline significant in individuals who ingested five different lactic acid strains in turn.

Consideration
Another study shows that helper T cells (CD4 ⁺ ) expressing CD25 molecules at high density (CD4 ⁺ CD25 ⁺⁺ ) are important in protecting against autoimmune diseases, allergies and inflammatory bowel disease It has been. This finding suggests that the number of these cells increases after ingesting different lactic acid bacteria in succession, and that individuals who are concerned about the risks associated with the progression of the disease may benefit from taking these bacteria. doing.

Example 3
animal
8-week-old C57BL / 6 mice were purchased from Taconic Europe (Denmark). The animals were raised and bred at a former animal facility at Dept. of Cell and organism biology. All experiments were performed according to the Ethics Committee of Marmo, Sweden. Mice were given regular food and water ad libitum. And at least one week before the start of the experiment, they were given a period of adaptation to the new environment. The mice used in the EAE experiment were weighed and examined in this experiment for clinical symptoms of EAE and scores according to the scale described in the daily clinical assessment. Score 6 mice received 0.5 ml of saline subcutaneously once a day to prevent dehydration. Food was placed at the bottom of the cage when clinical symptoms were confirmed in the mice. Score 7 mice were sacrificed for ethical reasons. The experiment was terminated 24 days after the start of immunization.

The encephalomyelitis-inducing peptide of the antigen myelin oligodendrocyte glycoprotein (MOG) was used to induce experimental autoimmune encephalitis (EAE). A synthetic peptide, amino acids 35-55 (MEVGWYRSPFSRVVHLYRNGK), was purchased from Schaefer N (Copenhagen, Denmark). The purity of the peptide used was 99%.

Immunity
EAE was induced in female C57BL / 6 mice as previously described. Briefly, each animal was immunized under isoflurane anesthesia by subcutaneous injection, and a CFA 1: 1 emulsion containing 200 μg MOG _35-55 and Mycobacterium tuberculosis H37Ra in PBS was added. μl was injected into the flank. Immediately thereafter, 48 hours after immunization, mice were injected intraperitoneally with 0.1 ml of 4 μg / ml pertussis toxin.

Clinical evaluation
Clinical signs of EAE were scored according to the following scale of 0 to 8. 0: No signs of clinical disease are seen. 1: weakened tail, 2: paralyzed tail, 3: hind leg paralysis and gait disturbance, 4: paralysis of one hind leg, 5: paralysis of both hind legs, and paralysis of the back of the torso, mice with front legs It can move around. 6: Paralysis of both hind legs, paralysis of the rear torso, and more serious mobility problems. 7: Paralysis of limbs, immobility, drowning, 8: death.

Bacterial strains and treatment
L. plantarum HEAL 9 and L. paracasei 8700: 2 were provided by Prove AB (Ideon, Lund, Sweden). Bacteria were harvested by centrifugation, washed once and then resuspended in tap water at a final concentration of 2 × 10 ⁹ colony forming units (CFU) / 100 ml with tap water. The third bottle was a mixture of L. plantarum HEAL 9 and L. paracasei 8700: 2, prepared at a final concentration of 2 × 10 ⁹ CFU / 100 ml. The bottles were washed and bacteria-containing vehicles were prepared daily. Experiments were performed with 10 mice in each treatment group. Each animal drank 4-5 ml vehicle containing about 10 ⁸ CFU per day.

result
Probiotic treatment is a probiotic treatment of three groups of animals to study the anti-inflammatory effects of probiotics that suppress the progression of MOG-induced EAE : L. plantarum HEAL9, L. paracasei 8700: 2 and L. Pre-treatment with a mixture of plantarum HEAL 9 and L. paracasei 8700: 2 for 12 days, followed by immunization for EAE with CFA and MOG _35-55 emulsion on day 0, followed by all experimental periods Treated through (through day 24). A fourth group of animals received only tap water as a control. As shown in FIG. 10, treatment with L. plantarum heel 9 or L. paracasei 8700: 2 was performed on day 10 compared to control mice showing severe EAE starting on day 11. Successfully prevented the progression of chronic EAE. Mice treated with the bacterial mixture also showed a delay of about 5 days of onset compared to controls. These mice progressed to more severe EAE compared to mice treated with a single bacterium. On day 24, the incidence of affected animals was significantly reduced, with 14% treated with a single bacterium versus 91% of control mice. In addition, the incidence was 60% in mice treated with the bacterial mixture. Analyzing the changes in the animal's body weight during the experiment, it was surprising to show that treatment with the bacterial mixture normally suppressed the weight loss that occurs after the onset of the disease (Figure 9). Animals treated with a single bacterium lost weight despite showing no symptoms of paralysis when compared to control mice.

Prophylactic treatment of EAE animals were treated with probiotics for 12 days before immunization and throughout the experiment.
100 ml of drinking water containing various bacteria was prepared fresh every day.
About 1.108 bacteria / mouse / day, control mice received water only.
N _Plamtarum = 7, N _Paracasei = 7, N _Mix = 10, N _Control = 11
The results are shown in FIG.

Therapeutic treatment of chronic inflammation in EAE
Oral treatment with probiotics one day after the onset of EAE. Animals were individually treated with a feeding needle.
About 1.108 bacteria / mouse / day. Control mice received 9 mg / ml sterile saline.
N _Plamtarum = 6, N _Paracasei = 6, N _Mix = 8, N _Control = 8
The results are shown in FIG.

Experimental Example 4
Inhibition of chronic inflammation of the central nervous system by oral administration of Lactobacillus paracasei and Lactobacillus plantarum The purpose of this study is to determine the effects of probiotics on T cell-mediated chronic inflammation in the central nervous system Was to consider. We hypothesized that oral treatment of probiotics provided an anti-inflammatory effect by immunomodulation of pathogenic T cell effectors.

Materials and methods Animal model Experimental autoimmune encephalomyelitis (EAE) is an animal disease model of multiple sclerosis, 35-55 of myelin oligodendrocyte glycoprotein (MOG) in complete Freund's adjuvant. Induced by immunization with the second peptide. Pertussis toxin was also injected in connection with further increased inflammatory effects. Two weeks later, the animals began to show clinical signs. EAE signs were scored into 8 categories. 0: No signs of clinical disease. 1: Tail weakened, 2: Tail paralyzed, 3: Paralyzed and gait disturbance, 4: One hind leg paralyzed, 5: Both hind leg paralyzed, 6: Both hind leg paralyzed and 3rd leg Paralysis, mouse can move forward. 7: Paralysis of limbs, immobility, drowning, 8: death.

Probiotic bacterial strains Lactobacillus paracasei 8700: 2, Lactobacillus paracasei defect, Lactobacillus plantarum HEAL 9, Lactobacillus plantarum HEAL 19, Lactobacillus plantarum 299v, Lactobacillus delbrueckii (Delbrueckii ) Was provided by Provider AB.

Treatment protocol Prophylactic treatment; 1.10 ⁹ to 1.10 100 ml of drinking water containing ¹⁰ different bacteria was prepared fresh daily. Each mouse drinks about 5 ml (about 1.10 ⁸ -1.10 ⁹ bacteria / mouse / day). Animals were treated for 7 to 14 days prior to immunization and during all experimental periods. Control mice received water only. In our recent experiments, animals are treated with a feeding needle on the 10th day after immunization every 2 days during the experimental period.
Therapeutic treatment; 10 days after onset, each animal received 1 × 10 ⁹ bacteria in individual treatment every 2 days during the experimental period. Control mice were given saline.
Methetraxate (MTX) was administered intraperitoneally at a final concentration of 2.5 mg / kg every 2 days during the experimental period.

Analysis Various organs and materials were collected from animals for further study: brain, spinal cord, spleen, lymph nodes (mesentery / groin), intestines (sites containing Peyer's plaques) and blood. Some organs were treated with isopentane for immunohistochemical analysis. Spleen and lymph node cells were analyzed by flow cytometry (FACS), or cultured for further cytokine ELISA studies and stimulated with MOG peptide, anti-CD3 and LPS.

Results Prophylactic treatment Animals were treated with 1.10 ⁸ Lactobacillus paracasei 8700: 2 or Lactobacillus plantarum HEAL19 for 7 days prior to immunization. EAE was significantly suppressed in a short period.

Animals were treated with 1.10 ⁹ Lactobacillus paracasei 8700: 2 or Lactobacillus plantarum HEAL 9 for 12 days prior to immunization. The onset of EAE was delayed for more than a week and the progression of EAE was slow (see Figure 11 and Figure 12)
Spinal cord immunohistochemical analysis showed significantly lower amounts of inflammatory T cells in probiotic-treated animals. An in vitro spleen cell proliferation assay revealed the same as MOG-specific proliferation of T cells, but interestingly, it produced significantly lower amounts of the inflammatory cytokines TNF-α and IFN-γ, and IL -4 and IL-10 produced higher amounts. Paracasei treated animals showed higher expression levels of total IgG and IgA antibodies in plasma. FACS analysis of mesenteric lymph nodes showed an increase in CD4 ⁺ CD25 ⁺ T cells. Immunohistochemical analysis of the spleen showed an increase in Foxp3 ⁺ (T _reg ) cells.
Screening 1:
Prophylactic treatment of 5 different lactic acid bacteria (Lactobacillus paracasei 8700: 2, Lactobacillus paracasei defecti, Lactobacillus plantarum Heal 9, Lactobacillus plantarum HEAL 19, Lactobacillus plantarum 299v) Screening was performed to suppress EAE by protocol.

Transfer 1;
Animals were treated for 2 weeks with Lactobacillus paracasei 8700: 2, Lactobacillus plantarum Heal 9, or a mixture of both strains. Mesenteric lymph node cells were harvested and transplanted into recipients (iv) immunized for EAE one day after transplantation. EAE was significantly suppressed by cells from animals treated with a mixture of the two strains. This inhibitory effect was eliminated by removing CD4 ⁺ cells (see FIG. 13).

  This experiment was repeated with a group of animals treated with a mixture of three strains including Lactobacillus paracasei 8700: 2, Lactobacillus plantarum Heal 9, Lactobacillus plantarum HEAL 19.

  The animals were immunized with EAE. Ten days after the onset of EAE, they were individually treated with a mixture of Lactobacillus paracasei 8700: 2, Lactobacillus plantarum Heal 9, Lactobacillus plantarum HEAL 19. Established EAE disease was significantly suppressed compared to controls that received saline. This experiment has succeeded in obtaining similar results even when repeated. Treatment with the non-specific bacteria Lactobacillus delbruecki or methotrexate (a common anti-inflammatory agent) did not show the unique therapeutic effect of the current protocol.

The anti-inflammatory mechanism of action of these therapies is not yet known, but our results clearly show an important role for the amount of CD4 ⁺ CD25 ⁺ Foxp3 ⁺ regulatory T cell population (see Figure 14) ).

FIG. 1 shows the number of volunteers who reported any minor side effects in the gastrointestinal system during the trial. FIG. 2 shows the baseline number of various lymphocytes per ml of blood (day 0) (mean ± (SEM)). FIG. 3 shows the percentage of baseline lymphocytes (Day 0) and GMFI (mean ± (SEM)) positive for various cell activation and memory markers. Subjects were randomly assigned to 9 different experimental groups. The trial was followed by a 2-week drug holiday followed by an active study period. During this period, subjects will receive one dose of study product per day for 14 days (L. plantarum Heal 19, L. fermentum, L. paracasei, L. gasseri, L. rhamnosus, P. runndensis group) or 35 Consumed daily (L. plantarum 299v and placebo group). Each dose is 10 ¹⁰ (lactic acid bacteria group) colony forming units (CFU) or 10 ⁹ CFU bacteria (P. runnsis group). The percentage of lymphocytes expressing the activated phenotypes CD8CD25, CD8HLA-DR, CD4CD25 and CD4HLA-DR was analyzed by flow cytometry. The group mean (± SEM) is based on the percentage of values within the individual, and is shown on the 14th / 0th day and the 35th / 0th day (L. plantarum 299v and placebo group only). The percentage of lymphocytes expressing the memory phenotypes CD8CD45RO and CD4CD45RO was analyzed by flow cytometry. The group mean value (± SEM) is based on the proportion of individuals, and is shown on day 14 / day 0 and day 35 / day 0 (only for L. plantarum and placebo groups). The percentage of lymphocytes positive for the NKT cell marker (CD56CD16CD3) was analyzed by flow cytometry. Group calculations are based on the percentage of individuals (day 14 / day 0). Neutrophil phagocytic activity was analyzed by incubating whole blood cells with FITC-labeled E. coli or S. aureus. The ratio of the average fluorescence value obtained on the 14th day and the 0th day was individually determined, and the group calculation values are shown in the figure. FIG. 9 shows prophylactic treatment, body weight, and clinical symptoms for the progression of EAE (Experiment 3). FIG. 10 shows the therapeutic treatment of chronic inflammation on the day of onset of EAE therapy after onset (Experiment 3). FIG. 11 and FIG. 12 show the delay in onset of EAE by giving L. paracasei 8700: 2 and L. plantarum HEAL9 compared to the control group. FIG. 11 and FIG. 12 show the delay in onset of EAE by giving L. paracasei 8700: 2 and L. plantarum HEAL9 compared to the control group. FIG. 13 shows that a mixture of the probiotic bacteria L. paracasei 8700: 2, L. plantarum HEAL 9 and L. plantarum HEAL 19 inhibits EAE. FIG. 13 further shows that this suppressive effect is disrupted by removing CD4 + T cells. FIG. 14 shows that a mixture of L. paracasei 8700: 2, L. plantarum / HEAL9, L. plantarum / HEAL19 inhibits EAE as compared to control, methotrexate, and L. delbruecki. FIG. 15 shows the percentage of lymphocytes expressing CD4CD25, an activated phenotype from experiment 2. FIG. 16 shows the percentage of lymphocytes expressing the activated phenotype CD4 ⁺ CD25 ⁺⁺ from experiment 2. FIG. 17 shows the percentage of lymphocytes expressing CD8 ⁺ HLA-DR ⁺ , an activated phenotype from experiment 2. FIG. 18 shows the percentage of lymphocytes expressing the activated phenotype CD8 ⁺ CD25 ⁺ from experiment 2. FIG. 19 shows the percentage of lymphocytes expressing CD4CD45RO, an activated phenotype from experiment 2.

Claims (10)

  Lactobacillus plantarum HEAL 9, DSM 15312, Lactobacillus plantarum HEAL 19, DSM 15313 for the manufacture of a pharmaceutical composition for the treatment and / or prevention of multiple sclerosis (MS) And use of at least one Probiotic bacteria strain of lactic acid bacteria selected from the group consisting of Lactobacillus paracasei 8700: 2, DSM 13434.   Use according to claim 1, wherein said at least two probiotic strains are used for the treatment and / or prevention of said multiple sclerosis.   Use according to claim 2, wherein said at least two strains are intended to be administered sequentially or simultaneously.   4. Use according to any one of claims 1 to 3, wherein the pharmaceutical composition is a liquid formulation or a solid formulation. Wherein the solid formulation is a tablet, suction tablets, Chi-menu queuing tablets (chewing tablet), mosquito capsule, sachets, powders, granules, coated particles and coated tablets, enteric-coated tablets and enteric capsules, melting strips And use according to claim 4, selected from the group consisting of: and films.   Use according to claim 4, wherein the liquid formulation is selected from the group consisting of oral solutions, suspensions, emulsions and syrups.   Use according to any one of claims 1 to 4, wherein the composition comprises a carrier material. Use according to any one of claims 1 to 7, wherein each said strain is present in the composition in an amount of 1 x 10 ⁶ to 1 x 10 ¹⁴ CFU. Use according to claim 8, wherein each said strain is present in the composition in an amount of 1 x 10 ⁸ to 1 x 10 ¹² . Use according to claim 9, wherein each said strain is present in the composition in an amount of 1 x 10 ⁹ to 1 x 10 ¹¹ .

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